Biological AgentsAnd

Factors Affecting Decontamination

Bruce R. Cords, Ph.D.Ecolab Inc.

B. anthracis Anthrax

Variola major Smallpox

Yersinia pestis Plague

Vibrio cholera Cholera

Filovirdia Ebola

Aphthovirus Foot & Mouth Disease

Botulinum Toxin Botulism

Biological Agents of Concern

MYCOBACTERIUM

PRIONS

BACTERIAL SPORES

PROTOZOA CYST/OOCYSTS(e.g. Crytosporidium)

NON-ENVELOPED VIRUSES

FUNGI

VEGETATIVE BACTERIA

LIPID-ENVELOPED VIRUSES

Resistance to Biocides

Relative Biocidal Activity

Peroxyacids, glutaraldehyde, formaldehyde, chlorine dioxide, ethylene oxide

Phenolics, Iodophors, Hypochlorites

Quaternary Ammonium Compounds, Organic Acids

Bacterial SporesMycobacteriumNon-enveloped Viruses

MycobacteriumNon-enveloped VirusesFungi

Vegetative BacteriaEnveloped Viruses

* There Are Exceptions!

Antimicrobial Tests(Required for EPA Registration)

Product Test Required Organisms

General disinfectant AOAC UseDilution

S. cholerasuis ATCC 10708S. aureus ATCC 6538

Hospital disinfectant AOAC UseDilution

S. cholerasuis ATCC 10708S. aureus ATCC 6538P. aeruginosa ATCC 1542

Sporicidal AOACSporicidal

B. subtilis ATOC 19659Cl. sporogenes ATCC 3584

Food Contact Surface SanitizerAOAC Germicidal Detergent Sanitizer Test

99 ml SanitizerUse-Solution25ºC

Add 1 ml of E.colior S.aureus

(minimum of 7.5 x 107

CFU/ml)

30 secondContactTime

Neutralize 1 mlEnumerateSurvivorsRequired

Efficacy:99.999% Kill in 30 seconds at 25ºC

0

15

30

Suggested Surrogates

Agent Surrogate Variola major (Smallpox) Vaccinia virus Yersinia pestis (Plague) Yersinia pseudotuberculosis Bacillus anthracis (Anthrax) B. subtilis B. cereus

B. globigii Foot-and-Mouth Disease Virus ? Norwalk Virus Feline calicivirus

Regulatory Hurdle

− EPA does not allow real or implied claims for any infectious agent that is not stated on the product label.

− Very few commercial products have been tested against the candidate biological agents.

Legal Issues

1) It is a violation of Federal law to use an EPA registered product in a manner inconsistent with its labeling.– Solution strength must be according to label– Applications must be on the label

2) It is a violation of Federal law for a manufacturer to make real or implied claims for efficacy against organisms which are not on the label.– Creates problems when we encounter:

• FMDV• Anthrax• Norwalk• SARS• Avian Influenza H5N1

Anthrax/ Bacillus anthracis− No products carry label claims (some have

crisis exemption)− Cutaneous, pulmonary, and

gastrointestinal transmitted by aerosols and on environmental surfaces

− Infectious dose - 5-10,000 spores (inhaled)− Effective agents (published literature)

– .25%-.50% (2500-5000 ppm) Peroxyacetic Acid– 2% (20,000 ppm) Glutaraldehyde– 4% (40,000 ppm) Formaldehyde– 1% (10,000 ppm) Sodium Hypochlorite

Smallpox/ Variola Major Complex Coat

− No products carry label claims− Transmitted through aerosols, contact

with infected person, and environmental surfaces

− Low infectious dose - a few virons− Effective agents (published literature)

– 1% (10,000 ppm) Sodium Hypochlorite– 2% (20,000 ppm) Glutaraldehyde– 2% (20,000 ppm) Formaldehyde

Levels of Decontamination Somewhat Dependent on Infectious Dose

Smallpox A few virons caninduce disease.

Anthrax Infectious dose(inhaled) may be10,000 spores

– Smallpox decontamination to 100 virons/m2 not acceptable

– Anthrax decontamination to 100 spores/m2 probably a safe level

Plague/ Yersinia pestis

− No products carry label claims− Transmitted as an aerosol through

respiratory droplets or plague-infected fleas. Organism does not survive more than a few hours outside the host.

− Infectious dose - 50-1500− Effective agents (published literature)

– 1% (10,000 ppm) Sodium Hypochlorite– 70% Ethanol– 2% (20,000 ppm) Glutaraldehyde– Many general disinfectants

Cholera/ Vibrio cholera

− No products carry label claims− Transmitted by contact and in water and

food contaminated with excreta from infection individuals

− Infectious dose 106

− Effective agents– General disinfectants

Ebola/ Filovirida Enveloped RNA Virus

− No products carry label claims− Transmitted by contact with infected

individuals or their blood/secretions. Environmental?

− Low infectious dose− Effective agents (published literature)

– 2% (20,000 ppm) Sodium Hypochlorite– 2% (20,000 ppm) Glutaraldehyde– .5% (5,000 ppm) Peracetic Acid– 1% (10,000 ppm) Formaldehyde

Foot & Mouth Disease/ Apthovirus

Non Enveloped RNA Virus− Some products carry Label Claims− Transmitted by aerosols, environmental

surfaces and direct contact between animals− Low infectious dose− Effective agents (published literature and

product approvals)– .03% (300 ppm) Peracetic Acid– .1% (10,000 ppm) Peroxymonosulphate– .05% (500 ppm) Sodium Hypochlorite– Other products (Ministry of Agriculture Fisheries

and Food; U.K.)

Is SARS a Potential Bioterrorism Agent?

1. Available? Yes2. Culturable? Yes3. Infective? Highly4. Deliverable? Unknown5. Decontamination? Likely to be

susceptible to common disinfectants

Lessons Learned from the Anthrax Case

1. We were not prepared for decontamination (e.g. Hart Building).

2) It took three treatments with ClO2 to achieve adequate results.

3) Many items were destroyed as opposed to decontaminated.

4) Ambulance chasers with “cure-alls” were abundant (e.g. spores vs. vegetative).

Bacillus anthracisSpores vs. Vegetative Cells

Peracetic Acid 5-logreduction

Spores 2,500 ppm for 30 min.

Vegatative 150 ppm for 30 sec.

Factors Contributing to Failure of Decontamination

Disinfectant/Biocide:

− Selection of biocide not effective against infectious agent

− Biocide too dilute− Insufficient contact time− Temperature too low*− Relative humidity too low

− (gaseous disinfectants)

Factors Contributing To Failure of Decontamination

Environmental Factors:

− Presence of organic matter*− Inactivation of QAC’s by residual soaps

and detergents− Incorrect application/coverage− Inadequate treatment of water supply− “Wettability” of surface

All Sanitizers Are NOT Created EqualReduced Temperature Sanitizer Efficacy

70oF. Well Water (250 ppm)30 Seconds

0123456

0.20% 0.35% 0.13% 0.26% AcidAnionic

Quat Iodophor Chlorine

S. aureus E. coli

Log

Reduction Peracetic Acid Organic Acid

All Sanitizers Are NOT Created EqualReduced Temperature Sanitizer Efficacy

55oF. Well Water (250 ppm)30 Seconds

0123456

0.20% 0.35% 0.13% 0.26% AcidAnionic

Quat Iodophor Chlorine

S. aureus E. coli

Log

Reduction Peracetic Acid Organic Acid

All Sanitizers Are NOT Created EqualReduced Temperature Sanitizer Efficacy

40 oF. Well Water (250 ppm)30 Seconds

0123456

0.20% 0.35% 0.13% 0.26% AcidAnionic

Quat Iodophor Chlorine

S. aureus E. coli

Log

Reduction

Peracetic Acid Organic Acid

0123456

0.20% 0.35% 0.13% 0.26% AcidAnionic

Quat Iodophor Chlorine

S. aureus E. coli

All Sanitizers Are NOT Created EqualReduced Temperature Sanitizer Efficacy

40 oF. Well Water (250 ppm)2 Minutes

Log

Reduction

Peracetic Acid Organic Acid

Effect of Temperature Organic Load and Concentration on Disinfectant

ActivityQuaternary Disinfectant:

(Log Reduction for P. aeruginosa)

20°C 10°Cx0.5 x1.0 x2.0 x0.5 x1.0 x2.0

.03% F P P F F PBSA

.3% F P P F F FBSA

P (Pass) = > 5 log reductionF (Fail) = < 5 log reduction

QUAT1000 ppm5 minutes

Taylor et al 1999

Effect of Temperature Organic Load and Concentration on Disinfectant

ActivityPeracetic Acid Disinfectant:

(Log Reduction for P. aeruginosa)

20°C 10°Cx0.5 x1.0 x2.0 x0.5% x1.0 x2.0

.03% P P P P P PBSA

.3% F P P F P PBSA

P (Pass) = > 5 log reductionF (Fail) = < 5 log reduction

Peracetic Acid300 ppm5 minutes

Taylor et al 1999

Treatment of a 48 HourBiofilm of Pseudomonas aeruginosa*

0

10

20

30

40

50

60

70

80 ppm 160 ppm 320 ppm

Peracetic/Organic Acid Peracetic Acid Chlorine

1 Minute Contact Time

*Fatemi and Frank. 1999. Journal of Food Protection. Vol. 62 (761-765)

Effect of Relative Humidity on Sporicidal Activity (B. globigii) of

Formaldehyde

LogConcentration RH Temp Reduction Time

400 mg/m3 30% 25°C 1 22’

400 mg/m3 98% 25°C 1 9’

Ref: Emerging Infectious Disease Vol 9 No 6 p. 625

Effect of Relative Humidity on Sporicidal Activity (B. subtilis v niger) of Peracetic Acid Vapor

LogConcentration RH Temp Reduction Time

1mg/L 80% RT >5 log 10’1mg/L 60% RT >4 log 10’1mg/L 40% RT >3 log 10’

Ref: Emerging Infectious Disease Vol 9 No 6 p. 626

Inactivation Of Bacillus spp. By Boiling In Tap Water

Initial log After 5’CFU/ml Boiling

C UNC C UNC

B. anthracis 4.95 4.92 0 2.01(sterne)

B. cereus 4.62 4.59 0 1.46(commercial)

B. cereus 4.54 4.76 0 0.48ATCC 9592

B. thuringensis 4.63 4.46 0 1.47ATCC 35646

C = Covered UNC = Uncovered

Temp above surface: C = 98.9°C UNC = 77.3°C

Ref: Emerging Infectious Disease Vol 10 No 10 2004

National Center for Food Protection and Defense

Study No: 910F2376745 – Ecolab

Development of Time/Temperature Concentration Matrix for Inactivation of Infectious Bioterrorism Agents by Chemical Biocides

Student: Hilgren – Ecolab

Co-Advisors: Swanson – EcolabDiez – University of Minnesota

General Method

Bacillus Spores

Food Soil• Egg Yolk Emulsion 8.8% protein

• Whole Milk 3.5% fat

• Flour Slurry 7.6% starch

Spore/soil mixture dried on stainless steel carrier

Carrier immersed in candidate biocide for 10 minutes

Impact of Food Soil on Inactivation of B. cereus spores by Peroxyacetic

Acid (Preliminary Results)

Conditions = 10 minute exposure20°CB. cereus ATCC 10987

Log Reduction5,000 ppm 10,000 ppm

No Soil >7.0 >7.0Whole Milk 1.5 4.0Egg Yolk 2.0 4.0Flour 2.0 7.0

Ref: Ecolab, NCFPD Project

Impact of Food Soil and Temperature on Inactivation of B. cereus Spores by

Peroxyacetic Acid (Preliminary Results)

Conditions = 10 minute exposure10,000 ppm Peroxyacetic acidB. cereus ATCC #10987

Log Reduction10°C 20°C 30°C

No Soil 6.0 >7.0 >7.0Whole Milk 1.5 4.0 >7.0Egg Yolk 2.0 4.0 5.5Flour 1.5 7.0 7.0

Ref: Ecolab, NCFPD Project

Impact of Food Soil on Inactivation of B. cereus spores by Sodium

Hypochlorite (Preliminary Results)

Conditions = 10 minute exposure20°CB. cereus ATCC 10987

Log Reduction @ 50,000 ppmNo Soil >7.0Whole Milk 1.2Egg Yolk 1.9Flour >7.0

Ref: Ecolab, NCFPD Project

Note: Flour @ 1,000 ppm - >7.0 log reduction

Other Results to Date:

− Quarternary Ammonium Chloride− Not effective at 75,000 ppm (7.5%), 20°C− Normal disinfection level = 200-800 ppm

− Iodophor− Not effective at 19,000 ppm (1.9%), 20°C− Normal disinfection level = 25 ppm

− Acidified Sodium Chlorite− Somewhat effective− Egg yolk and milk results not as good as flour

− Hydrogen Peroxide− Effective at 25-30%; 20°C− Not effective at 10°C

− Mixed Peroxyacid System− Results similar to peroxyacetic acid

Future Testing to Include:

− B. anthracis (nonvirulent strain)− Yersinia Pestis